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Colchicine antibodies - Principles of treatment and future

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1 Colchicine antibodies - Principles of treatment and future
B. Mégarbane, F.J. Baud Réanimation Médicale et Toxicologique, INSERM U705, Université Paris-Diderot, Hôpital Lariboisière, Paris, France

2 Introduction (1) History:
Colchicine is an alkaloid derived from Colchicum autumnale (Liliaceae family) - Native to temperate areas of Europe, Asia, and America - All poisonous plant, high concentrations in the corm or bulb History: - Ephemeron (Theophrastlus of Eresus, 370 BC ) - Destructive fire of Colchican Medea (Nicander, 150 BC) - Treatment of gout (Alexander of Tralles, 550 AD) - Chemical synthesis (Laborde & Houdé, 1884)

3 Introduction (2) Indications: Gouty arthritis
Familial Mediterranean fever Scleroderma, amylmoidosis, Behcet’s disease, Paget’s disease Dermatitis herpetiformis, Acute neutrophilic dermatosis Erythema nodosum leprosum, Condyloma acuminata Resistant idiopathic thrombocytopenic purpura Biliary and liver cirrhosis

4 Acute colchicine poisoning
• Rare in Western countries • Resulting from both accidental and intentional overdoses • Responsible of a high-rate mortality 51 None 31 Minor 19 Moderate 3 Major 4 Deaths 323 case mentions 208 single exposures AAPCC-TESS, Clin Tox 2006 355 exposures in 33 countries in Europe during with 4% fatal rate Kupferschmidt H, EAPCCT 2005 Colchicine toxicity is predictable and dose-dependent with multiorgan involvement and delayed onset.

5 Mechanisms of colchicine action
• Selective and reversible binding to the 65 subunits of microtubules - In vitro : high affinity for dividing cells, mitotic apparatus, cilia, sperm tails and brain - Forensic studies: cell blockage in metaphase and abnormal nuclear morphology • Alteration in multiple cellular functions: - Cell shape, mobility, ability to phagocytosis - Cell division • Affection of organs exhibiting the highest cell turnover (GI mucosa, bone marrow) • Other effects: -  collagenase activity -  production of PG-B and PG-F2 in synovial cells -  transport of nucleosides

6 Toxicokinetics of colchicine
Conflicting data ... • A narrow therapeutic index • Rapid absorption with variable bioavailability ( %) • Hepatic metabolism (deacetylation), biliary secretion, enterohepatic cycle • Renal elimination (20%, unchanged form) • Role of co-ingested drugs (CYP3A4 inhibitors, macrolides) Bi-exponential curve Elimination half-life : hrs Distribution volume : 21 l/kg Rochdi M. Hum Exp Toxicol 1992

7 Colchicine poisoning Signs are well documented, follow a typical pattern, and involve multiorgan systems Stage I (0-12 h) GI symptoms Volume depletion Peripheral leukocytosis Stage II (2-7 d) Respiratory distress, ARDS, hypoxemia Cardiovascular shock Thromboyctopenia, DIC Myelosuppression, neutropenia Hyponatremia, hypocalcemia, hypophosphatemia Metabolic acidosis Rhabdomyolysis, myoglobinuria, oliguric renal failure Stage III (1-2 wk) Rebound leukocytosis Alopecia.

8 Experimental assessment of cardiotoxicity
Impairment of the intrinsec contractility of rat left ventricular papillary muscle (2 and 4mg/kg colchicine versus saline) • Impairment of myocardial contractility:  maximum shortening velocity (-32% and -61%)  active isometric force (-47% and -65%)  peak power output (-57% and -69%) • Impairment of isotonic relaxation and load dependence of relaxation, suggesting a decrease in sarcoplasmic reticulum function • Acceleration of isometric relaxation, suggesting a decrease in Ca++ myofilament sensitivity • Marked negative inotropic effects if impairment of myothermal economy Mery P. Intensive Care Med 1994

9 Colchicine cardiac toxicity
Cardiovascular and direct negative inotropic effects were largely assessed. 2 series with very elevated mortality rate: • Bismuth C. Presse Med 1977: 11 cases ≥ 0.8mg/kg GI troubles + DIC + aplasia + cardiac shock = 100% death • Sauder P. Hum Toxicol 1983: 8 cases mg, 4 cases with cardiac failure ( cardiac index,  systemic resistance) = 100% death Few cases report with survive despite cardiogenic shock: • Baron DA. Ouest Med F 38 yrs, 30 mg, WBC 27,000 /mm3, FV 22% • Bismuth C. Nouv Presse Méd M 37 yrs, 0.8 mg/kg, WBC 12,000 /mm3, PT 19% • De Villota E. Crit Care Med M 50 yrs, 30 mg, WBC 9,100 /mm3

10 Assessment of colchicine mechanism of cardiac toxicity
Sauder P. Hum Toxicol, 1983

11 T-waves negativation, ST elevation in leads I, II V2-V6
Profound EKG changes : Colchicine may impair impulse generation and cardiac conduction, in addition to the electrolyte and acid-base disturbances Cardiac dysrhythmias (sinus tachycardia, sinus bradycardia, VF, sinus arrest, complete AV blockade) T-waves negativation, ST elevation in leads I, II V2-V6 Stahl. Am J Med Sci 1979 Stapczynski. Ann Emer Med 1982 Hobson. Anaesth Intensive Care 1986 Wells. Vet Hum Toxicol 1989 Stemmermann. Hum Pathol 1972 McIntyre. J Forensic Sci 1994 Weakley-Jones. Am J Forensic Med Pathol 2001 Wells. Vet Hum Toxicol 2000 Brvar. Wien Klin Wochenschr 2004 Murray. Mayo Clin Proc 1983 Mendis. Postgraduate J 1989 Wells. Vet Hum Toxicol 1989 Mullins. Am J Emerg Med 2000 Weakleyy-Jones. Am J Forensic Med Pathol 2001 Brvar. Crit Care 2003 Miller. J Emerg Med 2005 Van Heyningen C. Emerg Med J 2005

12 Direct colchicine-related cardiac injuries
• Biology alterations: Delayed elevation of myocardial enzymes (troponine, CPK-MB) Mullins ME. Am J Emerg Med 2000 Sussman JS. Ther Drug Monit 2004 Van Heyningen C. Emerg Med J 2005 •  Pathology: a series of 12 fatal cases Interstitial edema without cell necrosis in all cases. Interstitial myocarditis in 2 cases. Hoang C. Ann Pathol 1982

13 Prognostic factors • The supposed ingested dose Mortality
< 0.5 mg/kg < 5 % 0.5 à 0.8 mg/kg % > 0.8 mg/kg 90 % • Prothrombin index ≤ 20% • WBC ≥ 18x109 /l • Onset of cardiogenic shock • Onset of ARDS in the 24th hours in the 72th hours Bismuth C. Presse Med 1977

14 Colchicine poisoning management
• Management includes early GI decontamination, careful monitoring of physical examination and laboratory tests (electrolytes, blood gases, appropriate cultures) and supportive treatments: • To date, there is no successful commercially available specific therapy, although several experimental studies and one human case report assessed the efficiency of colchicine-specific Fab fragments. Fluids (diarrhea) Vasopressors (shock) Oxygen supplementation, mechanical ventilation (ALI, ARDS) Antibiotics (fever) GCSF (neutropenia) Transfusions (thrombocytopenia, anemia)

15 Principles of immunotoxicotherapy
• A procedure able to simultaneously sequester, extract or redistribute, and eliminate the toxin by using specific active binding sites derived from different antibody molecular entities. • Currently used in humans for treating cardiac glycoside and venom poisonings. Since the initial report in 1976 by Smith et al., it has become the first-line treatment of life-threatening intoxication by cardiac glycosides, including digoxin, digitoxin, and other structurally related cardiotoxins from Nerium, Thevetia sp. (oleander), and Bufo sp. (toads). Scherrmann JM. Clin Toxicol 1989

16 Specific active biding sites derived from antibody
• Industrial manufacturing • Polyclonal antibodies of ovine origin • Fragmentation of antibodies: - useful for haptens (< 1000 d) - increased safety - limited instability • Future trends: monoclonal, humanized, semi-synthetic, further fragmentation, ...

17 Toxin sequestration Pharmacokinetic characteristics: Affinity issues:
•  Vdtoxin/Vdantitoxin ratio  1 to  probability of interaction For toxins with vascular distribution: IgG, IgM (5 l) For toxin with extravascular distribution: Fab (30 l) • Equilibration time in the distribution space: 2-4h (Fab) versus h (IgG, Fab’2) Affinity issues: • Affinity ≥ 109 M-1 (critical minimal value) to form stable complexes • Prefer polyclonal to monoclonal antibodies, based on manufacturing capacity + to enlarge specificity to epitopes. • Prefer ovine to equine antibodies, to reduce serum sickness + to produce a higher proportion of specific IgG

18 Toxin extraction or redistribution
Involve the removal of toxin from the toxin receptor compartment to the antibody distribution space Limiting factors: - Reversibility of binding (reversible intracellular binding) - Kinetics of the toxin release from the receptor t1/2 dissociation of colchicine / tubulin : 20 h t1/2 dissociation of digoxin / Na,K-ATPase: 1 h Blood anti-toxin concentration should exceed bound toxins during a predicted period of time to allow toxin efflux Anti-toxin with slow clearance (IgG, Fab ’2 < Fab) should be preferred to allow redistribution over a prolonged period

19 Toxin elimination Low MW-toxin bound to anti-toxin adopt anti-toxin elimination properties Extra-renal clearance: = 60-70% Reticulo-endothelial tissues gut, liver, spleen, lymph nodes Renal clearance: • Fab are filtrated through glomeruli, rapidly and extensively reabsorbed and catabolized by the proximal tubule cells. • Fab increases the renal clearance of toxins: % However: Possible tubular re-absorption Re-circulation of free toxin molecules Reduction in glomerular filtration 

20 Pharmacokinetic aspects of basic mechanisms
Phase 1 Phase 2 Phase 3 Total toxin level Free toxin level Fab level

21 Developmental requisites for immunotoxicotherapy
Colchicine is a good candidate for the development of a successful immunotherapy, as it perfectly answers to the toxin-dependent requisites. 1- Severe poisonings with a high risk of death and short-term effects 2- Toxicity in the milligram range, allowing stoechiometric neutralization 3- Efficient production of antibody in animals after conjugation to a protein 4- High affinity antibody ( M-1) 5- Distribution volume much greater than its corresponding Fab with the possibility of a rapid redistribution from tissue to blood

22 Limitations in colchicine poisoning (1)
Colchicine characteristics may theoretically limit the potential benefits Whereas cardiac glycosides are membrane-associated toxins, efficiently reversed with an equimolar dose of specific Fab colchicine is low-MW acting intracellular poison, needing to consider an expanding immunotherapy model to intracellular toxins.

23 Limitations in colchicine poisoning (2)
By opposite to digitalis poisonings where only one organ, the heart, is at vital risk, severe colchicine poisonings induce multi-organ failure with not always reversible structural injuries. • There is no evidence that antibodies can reverse organ injuries. • In order to consider any therapeutic interest of antibodies, it is necessary to hypothesize that neutralization of residual unbound colchicine may convert a lethal intoxication into a disease compatible with survival, even if a greater part of the toxin has already damaged the organism.

24 Assessment of the efficacy of toxin detoxification using immunotoxicotherapy
To test the reversal of toxicity To test the sequestration effect To test the best fragments and administration conditions Scherrmann JM. Clin Toxicol 1989

25 In vitro experimental studies (1)
Partial reversion of colchicine-induced inhibition of tubulin polymersiation using colchicine-sepcific antibodies Wolf AD. J Biol Chem 1980

26 In vitro experimental studies (2)
Colchicine neutralization with antibodies has proven to be effective. Although intracellular binding of colchicine to microtubules was expected to limit immunotoxicotherapy efficacy, a reversible effect on microtubules was found. • Colchicine-induced polyploidy and chromosomal aberrations in a model of Chinese hamster ovary cell were reversible with a specific high-affinity monoclonal antibody, even when administered up to 6 hours after colchicine exposure Rouan SE. Am J Pathol 1990  • A tightly bound intracellular toxin was extracted with high-affinity antibodies at a rate depending on its dissociation rate from its receptors. Kinetics was of first-order decline with t1/2 ranging from 15.5 to 16.4 h Chappey ON. J Pharmacol Exp Ther 1995

27 In vivo experimental studies (1)
Anti-colchicine active immunization of rabbits: - Protective effects against 3-mg/kg colchicine (> LD50), with an antibody titer-dependent response. - Effective trapping of colchicine Biexponential - t1/2: 12h Unbound colchicine < 0.5% Scherrmann JM. Clin Toxicology 1989

28 In vivo experimental studies (2)
In mice, colchicine-specific IgG administration, even after colchicine distribution phase, significantly decreased mortality rate. Terrien N. Toxicol Appl Pharmacol 1990 In mice, colchicine-specific goat IgG (1/2-1/8 molar dose) favorably improved outcome when previously (90 min) receiving IP 3.8-mg/kg colchicine lethal dose. Sabouraud AE. Toxicology 1991

29 Colchicne Fab fragments-related alteration in kinetics
Tissue extraction & elimination: In mice: Fab induced • Decrease in colchicine VD • Colchicine sequestration in intravascular spaces • Decrease of colchicine concentrations in most tissues Sabouraud AE. J Pharm Pharmacol 1992 In rabbits: Fab infusion 1.5 h after 0.1-mg/kg colchicine, over 0.25 h, 1/2-molar dose Colchicine concentration x10-16 within 15 min Total plasma AUC x20 Undetectable free plasma fraction over a period of 2 h • Decrease of the VD /24 • Decrease of total clearance: /17 • Reduction of biliary excretion: - 80% • Reduction of the dose fraction excreted in urine: 9% versus 38% Sabouraud AE. J Pharmacol Exp Ther 1992

30 Case report of a severe human colchicine overdose treated with colchicine-specific Fab fragments (1)
25-year-old woman, suicidal attempt, 0.96 mg/kg (60 mg) colchicine. - Call: 24 h after ingestion due to severe GI pain - On the scene: HR 110 /min, unrecordable BP 500 ml colloid and 10 mg/kg/min dobutamine - ICU: °C, BP 110/80 mmHg, HR 110 /min, RR 60 /min, Creatinine 140 µmol/l, WBC 69,300 /mm3, platelet 268,000 /mm3, PT 14%, V <5% pH 7.38, PaCO2 28 mmHg, PaO2 86 mmHg (O2 3 l/min) Chest X-Rays: pulmonary edema Plasma colchicine: 24 ng/ml (RIA) Gastric lavage + activated charcoal Crystalloid (4400 ml) and colloid (1100 ml) Deterioration of hemodynamic status despite dobutamine Baud FJ. N Engl J Med 1995

31 Case report (2) -45 min -15 min +1H +12H PAP (mmHg) 23 18 13 11
PCWP (mmHg) SBP (mmHg) HR (/min) Cardiac Index (l/min/m2) Resistance ( AV difference in O Plasma lactate (mmol/l) ND Dobutamine (µg/kg/min) Dopamine (µg/kg/min) Norepinephrine (mg/h) Baud FJ. N Engl J Med 1995

32 Infusion of goat colchicine-specific Fab fragments H36 after ingestion
Case report (3) Infusion of goat colchicine-specific Fab fragments H36 after ingestion • Antiserum derived from goats immunized with a conjugate of colchicine + serumalbumine • Affinity: 2 x 1010 M-1 • No separation of colchicine-specific Fab fragments (7.5%) from others • Dosage: 480 mg (6.4 g Fab fragments) in 160 ml 240 mg over 1-hour period 240 mg over 6 hours Baud FJ. N Engl J Med 1995

33 Case report (4) -45 min -15 min +1H +12H PAP (mmHg) 23 18 13 11
PCWP (mmHg) SBP (mmHg) HR (/min) Cardiac Index (l/min/m2) Resistance ( AV difference in O Plasma lactate (mmol/l) ND Dobutamine (µg/kg/min) Dopamine (µg/kg/min) Norepinephrine (mg/h) Baud FJ. N Engl J Med 1995

34 Case report (5) Toxicodynamics:
• Significant arterial vasodilatation within 30 min. • Marked improvement in all indexes of tissue perfusion. • Withdrawal of inotropic drugs within 36 h. • ARDS took longer to subside. •  No prevention of the delayed occurrence of bone marrow aplasia, complete hair loss, and transient peripheral neuropathy. • No adverse effect (hypersensitivity, serum sickness) or recurrence of toxic signs Survival with the well-known sequential phases however with a shortened duration Baud FJ. N Engl J Med 1995

35 Case report (6) Toxicokinetics:
•  total plasma concentration (x6: 12 to 122 ng/ml), as soon as 10 min after infusion, whereas free concentration became undetectable •  urinary excretion (x6), initially bound to Fab fragments Renally-excreted colchicine: 5.2 mg • Neutralized colchicine = 3.7 mg / 9 mg of present colchicine •  Fab/total colchicine ratio: 4 (0-1 h), 2 (1-7h) and <1 (>7h) T1/2: 25 h Baud FJ. N Engl J Med 1995

36 Case report (7) The patient’s clinical improvement was explained by the direct effects of colchicine-specific Fab fragments: • Redistribution from tissues into plasma Increase in plasma concentration • Sequestration into plasma compartment High affinity of Fab to colchicine • Immunoneutralization of the effects Decrease of protein-unbound colchicine (during 7h-infusion) and subsequent partial rebound (H12) Baud FJ. N Engl J Med 1995

37 The situation today? To date, no commercial preparation of colchicine-specific Fab fragments is available. Why ? Solved problems: • Specific antibody development is difficult • Fragmentation technique is a complex procedure • Large amount of fragments is required for a stoechiometrical neutralization Unsolved problems: - Inadequation between development costs and potential prescriptions (orphan) - Difficulties (legislation and bureaucracy) to organize an European trial

38 Further considerations for colchicine immunotoxicotherapy
Regarding the indications and time to administer: • Immediate or potentially life-threatening intoxication • Concept of critical dose (when a risk of mortality occurs) • Challenge the classical thinking of the necessity of rapid administration of the total Fab dose to improve poisoning. Regarding the minimal efficient dose to administer: • Optimal amount to neutralize = total ingested dose - critical dose • Inframolar neutralization may be efficient as in animal reports. • Removal of a modest portion of colchicine may dramatically improve outcome. Regarding the therapeutic action: Further studies are needed to determine the Fab effect on bone marrow aplasia. Baud FJ. Arch Toxicol Suppl 1997

39 Conclusions: • Coclchicine-specific Fab fragments may be useful to complete the supportive care in the most severe colchicine poisonings. • However, to date, these fragments are still not commercially available. • Until issues of cost and supply are worked out, coclchicine-specific Fab fragments remain a dream in the areas where poisonings are frequent and specific therapy urgently required.

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